Project Summary A key method to gather data from dynamic neural networks in living model organisms is two-photon microscopy together with genetically-encoded fluorescent proteins. There is a need to optimize fluorescent proteins for the two-photon regime, as some desirable characteristics (e.g. brightness) do not translate from one-photon optimization. This project aims to fill that gap with the directed evolution of a two-photon bright green fluorescent protein. Multiple constructs containing this fluorescent protein will be created for use in two-photon imaging. The directed evolution will be implemented using a new optical setup that can collect two-photon excited fluorescence from individual E. coli colonies on an agar plate. The brightest mutants from ~8 rounds of evolution will be crystallized, and one will be made into a tandem dimer. The dimer will be incorporated into four different constructs: 1) with a DREADD receptor, 2) with a channelrhodopsin, 3) in a histone H2B protein fusion, and 4) in a Cre-FLEX-switch expression vector. All constructs will be tested in HEK cells.